Expression in yeast of antigenically active, recombinant hybrid glutamic acid decarboxylase

ABSTRACT

An antigenically active hybrid glutamic acid decarboxylase (GAD) comprising an amino-terminal moiety derived from the GAD67 isoform linked directly or indirectly with a middle and carboxy-terminal moiety derived from the GAD65 isoform, and production thereof as a recombinant protein by expression in eukaryotic host cells, particularly yeasts.

FIELD OF THE INVENTION

This invention relates to the production of an antigenically activehybrid glutamic acid decarboxylase (GAD) molecule as a recombinantprotein by expression in eukaryotic host cells, particularly yeasts, andto the use of this recombinant hybrid GAD in the diagnosis andpresymptomatic detection of insulin-dependent (Type 1) diabetes mellitus(IDDM). The invention also relates to the use of this hybrid GADmolecule to inhibit or prevent the occurrence of IDDM in individuals whoare diagnosed or detected as having presymptomatic IDDM.

BACKGROUND OF THE INVENTION

Insulin dependent diabetes mellitus (DDM) is an autoimmune disease inwhich there is a characteristic immunological reactivity to a limitedset of tissue-specific cytoplasmic autoantigens of pancreatic islet betacells. Reactivity to one of these autoantigens, glutamic aciddecarboxylase (GAD; EC 4.1.1.15), is virtually unique to the disease,the rare exceptions being the neurological disorder, Stiff man syndrome(Solimena et al., 1990; Baekkeskov et al., 1990), and the polyendocrinesyndrome Types 1 and 2. Glutamic acid decarboxylase catalyses theconversion of L-glutamic acid to γ-aminobutyric acid (GABA) and carbondioxide (Erlander et al., 1991). GABA is a major inhibitoryneurotransmitter, and hence most research on GAD until recently hadconcentrated on the role of this enzyme in neural functioning. A newdirection developed with the recognition that antibodies to GAD areprevalent in IDDM (Baekkeskov et al., 1990).

GAD exists as 2 isoforms denoted by their calculated molecular weightsas GAD65 and GAD67. These differ according to their subcellular location(Erdo and Wolff, 1990; Faulkner-Jones et al., 1993), chromosomallocation (Erlander et al. 1991; Karlsen et al., 1991; Bu et al., 1992;Michelsen et al., 1991), amino acid sequence (Bu et al., 1992) andcofactor interactions (Erlander and Tobin, 1991) but have closehomology, in man 65% identity and 80% similarity. The greatestdivergence between the isoforms occurs in the first 100 amino acids (Buet al. 1992). The availability of cDNA clones encoding the two GADisoforms has allowed the expression of these in various systems,including bacteria (Kaufman et al., 1992), Sf9 insect cells using thebaculovirus vector (Seissler et al., 1993; Mauch et al., 1993), COS7monkey cells (Velloso et al., 1993), baby hamster kidney cells (Hagopianet al., 1993), yeast (Powell et al, 1995) and by in vitro translationusing rabbit reticulocyte lysate (RRL) (Petersen et al., 1994; Ujiharaet al., 1994; Grubin et al., 1994). In addition, a modified GAD65without the hydrophobic amino acids 245 inclusive of the N-terminalregion has been expressed in yeast (Powell, et al., 1996).

The identification of GAD as a major autoantigen of IDDM has led to theextensive use of this antigen in immunoassays for the accurate diagnosisand prediction of IDDM in at-risk populations. Such studies have shownthat antibodies to GAD are detectable in patients up to 10 years beforethe early onset of clinical symptoms (Baekkeskov et al., 1987, Atkinsonet al., 1990; Rowley et al., 1992; Chen et al., 1993; Tuomilehto et al.,1994; Myers et al., 1996). These assays have employed autoantigenic GADderived from two major sources. One source is animal materials, mostcommonly porcine brain, purified by affinity chromatography, andlabelled with radioactive iodine. The other source is in vitrotranscription and translation of the cloned human GAD65 gene, usingrabbit reticulocyte lysate (RRL) which produces biosyntheticallylabelled GAD suitable for radioimmunoprecipitation (RIP) assays(Guazzaroti et al., 1995). Expression from RRL has been widely used indiagnostic assays for anti-GAD in human sera but the in vitro expressionsystem has limitations in that only very small amounts, in the order ofpicomoles, of GAD are produced and the process is very costly. Bacterialexpression does not appear to yield GAD that is amenable to use indiagnostic assays, and yields from mammalian cells are unsuitably low.Several authors have presented evidence that the GAD must be in aparticular conformation to be reactive with antibodies in IDDM sinceIDDM sera generally do not show reactivity with GAD in Western blottingunder denaturing conditions, yet show potent reactivity undernon-denaturing conditions (Rowley et al., 1992; Tuomi et al., 1994,Myers et al., 1996); the GAD conformation that is recognised by themajority of IDDM sera is sensitive to exposure to reducing agents suchas β-mercaptoethanol, since GAD that has been thus treated losesreactivity (Tuomi et al., 1994). It is also recognised that antibodiesto GAD65 in IDDM react with particular epitopes on the molecule that liein the mid-region and C-terminal region of the molecule. Fusions ofcDNAs that encode particular sequences of GAD65 and GAD67 have beencreated as "chimeric" proteins to establish epitope recognition (Daw andPowers, 1995).

Assays using immunoprecipitation and either affinity purified porcinebrain GAD (Rowley et al., 1992) or recombinant GAD (Kaufman et al.,1992; Seissler et al., 1993; Mauch et al., 1993; Velloso et al., 1993;Hagopian et al., 1993; Petersen et al., 1994; Ujihara et al., 1994;Grubin et al., 1994) have revealed that 70-80% of IDDM sera containautoantibodies to GAD65, whereas only 8-25 % contain antibodies toGAD67. The autoantibodies in Stiff-man syndrome react with GAD byimmunoblotting under reducing conditions (Solimena et al., 1990),whereas autoantibodies to GAD in IDDM seldom do so (Baekkeskov et al.,1990), and are thought to recognise a conformational epitope (Tuomi etal., 1994). Epitopes have been mapped by examining the reactivity byimmunoprecipitation of IDDM sera against truncated polypeptides ofGAD65, with one major IDDM associated epitope located to the middle andcarboxy terminal domains of GAD65 (Kaufman et al., 1992; Richter et al.,1993; Ujihara et al., 1994). More recent mapping for epitopes foranti-GAD suggests the presence of two discontinuous epitopes, one withinamino acids 244 to 433, and the other within amino acids 451 to 570 (Dawand Powers, 1995). Curiously, whilst antigenically active regions ofGAD65 and GAD67 are highly homologous, GAD65 is the isoform with whichautoantibodies are predominantly reactive.

The further development of diagnostic assays for IDDM would greatlybenefit from a more accessible source of large amounts of recombinantGAD that would be free of the possible biohazards associated withmammalian sources. Furthermore, the search for disease-associatedepitopes of GAD would be facilitated by the availability of a simplesystem with which to carry out site-specific mutagenesis and deletionstudies. The requirement for GAD to be in a particular conformation hashindered prior efforts to use recombinant DNA technology to producelarge quantities of antigenically active GAD. Although the use of E.coli expression systems is reported to produce enzymatically active GAD(Kaufman et al., 1992), published reports on the utility ofbacterially-expressed antigenically active GAD for immunoassays arescarce and, as stated above, it is the experience of the presentinventors that this material performs less effectively inradioimmunoprecipitation tests with IDDM sera than does GAD expressed inother systems. The preferred expression system should be capable ofproducing not only large amounts of GAD, but also GAD that is in anappropriate conformation to be recognised by IDDM sera.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a hybrid glutamic aciddecarboxylase (GAD) which comprises an amino-terminal moiety derivedfrom the GAD67 isoform linked directly or indirectly with a middle andcarboxy-terminal moiety derived from the GAD65 isoform.

This hybrid molecule, which may also be referred to as a chimericmolecule, is referred to herein as "hybrid GAD67/65". The generation ofthis hybrid molecule is based on the reported location of autoimmuneepitopes in the middle and carboxy-terminal moieties of GAD65, andobservations by the present inventors that the amino-terminal moiety ofthe cDNA of GAD67 is more amenable than the cDNA of GAD65 to insertioninto vectors appropriate to expression of recombinant proteins.

As used herein, references to an "amino-terminal moiety" refer to amoiety comprising amino acid 1 to amino acid 90-105, more particularlyamino acid 1 to amino acid 95-101. Similarly, references herein to a"middle and carboxy-terminal moiety" refer to a moiety comprising ofamino acid 90-105 to amino acid 585, more particularly amino acid 95-101to amino acid 585. In the particularly preferred embodiment of thepresent invention, the hybrid GAD67/65 molecule comprisesGAD67(1-101)/GAD65(96-585).

Preferably, the amino-terminal GAD67 moiety is fused or linked directlyto the middle and carboxy-terminal GAD65 moiety, however these moietiesmay optionally be linked indirectly through a linker moiety of from 1 to50, preferably from 1 to 20, and more preferably from 1 to 5, amino acidresidues.

The hybrid GAD67/65 molecule of this invention may also comprise othermoieties fused or otherwise coupled thereto at either end of themolecule, for example moieties to assist in purification of the hybridGAD67/65 molecule when produced as a recombinant protein, such as aglutathione-S-transferase (or GST) moiety, a β-galactosidase moiety, ora hexa-His moiety.

In another aspect, the present invention provides an isolated nucleicacid molecule, preferably a DNA molecule, comprising a nucleic acidsequence encoding a hybrid GAD67/65 molecule as broadly described above.Preferably, the sequence comprises a fusion of cDNA sequences whichencode GAD67 (1-101) and GAD65 (96-585).

Such a nucleic acid molecule may comprise a recombinant DNA molecule, arecombinant DNA cloning vehicle or vector, or a host cell, preferably aeukaryotic host cell, comprising a nucleic acid sequence encoding thehybrid GAD67/65 molecules.

The present inventors have found that the hybrid GAD67/65 molecule canbe expressed in a bacterial (E. coli) system to provide good yields ofenzymatically active GAD, but immunoreactivity of the bacteriallyexpressed hybrid GAD67/65 is not ideal. Accordingly, expression in aeukaryotic host cell, particularly a yeast species such as Saccharomycescerevisiae, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha,or Schizosaccharonyces pombe, is preferred.

Thus, in a preferred aspect, the present invention provides a method forthe preparation of the hybrid GAD67/65 molecule which comprisesexpression of a nucleic acid sequence encoding the hybrid GAD67/65molecule in a eukaryotic host cell, particularly a yeast cell such as S.cerevisiae, and recovery of the expression product.

The invention also extends to recombinant hybrid GAD67/65 produced byexpression in a host cell, particularly in a yeast or other eukaryotichost cell, as described above.

Suitable expression control sequences an d host cell cloning vehicle orvector combinations for expression of recombinant proteins in eukaryotichost cells, particularly in yeasts such as S. cerevisiae and othereukaryotic host cells, are well known in the art, and are described byway of example in Sambrook et al. (1989) and by Sudbery (1996).

Recombinant hybrid GAD67/65 in accordance with the present invention maybe used advantageously in place of purified native or recombinant GAD65in the diagnosis and presymptomatic detection of IDDM in humans ornon-human mammals using assay techniques previously described.

Accordingly, in yet another aspect, the present invention provides amethod for the diagnosis and presymptomatic detection of IDDM in apatient characterised in that hybrid GAD67/65 as broadly described aboveis used to detect autoantibodies to GAD in a serum or other sample takenfrom the patient.

In a further aspect, the present invention provides a method oftreatment to inhibit or prevent the occurrence of IDDM in a patienthaving presymptomatic IDDM, which comprises administration to thepatient of an effective amount of hybrid GAD67/65 as described above.Preferably, the hybrid GAD 67/65 is administered by the mucosal route,most preferably by oral administration.

This method of treatment may be used to inhibit or prevent IDDM whendetected in a patient in a preclinical state (i.e. presymptomatic IDDM)by immunoassays for autoantibodies or other diagnostic methods. Broadly,the objective of the treatment is to re-establish normal immunetolerance and thereby abrogate the autoimmune process that gives rise toIDDM. Accordingly, references herein to "inhibit" or "inhibition", or to"prevent" or "prevention" are intended to refer to modulation of thestate of autoimmunity affecting pancreatic islet cells of the patient byadministration of the hybrid GAD67/65 in such a way and under suchconditions as to reinduce a state of immune tolerance ("tolerogenesis")to autoantigenic constituents of pancreatic islet cells.

As used throughout this specification, the term "patient" includes bothhumans and non-human mammals. Preferably, the patient is a human. It isto be understood, however, that the diagnostic and therapeutic methodsof the present invention are also applicable in non-human mammals suchas livestock, companion animals, laboratory test animals and captivewild animals. It is to be understood that livestock animals encompassanimals such as horses, cattle, sheep, goats, donkeys and pigs,companion animals include dogs and cats of all varieties, laboratorytest animals include mice, rats, guinea pigs and rabbits, and caputredwild animals include for example monkeys, kangaroos etc.

In yet another aspect, the present invention provides the use of hybridGAD67/65 as broadly described above, in the manufacture of apharmaceutical or veterinary composition for treatment to inhibit orprevent the occurrence of IDDM in a patient having presymptomatic IDDM.

The invention also provides a pharmaceutical or veterinary compositionfor use in treatment to inhibit or prevent the occurrence of IDDM in apatient having presymptomatic IDDM, which comprises hybrid GAD67/65 asbroadly described above, together with one or more pharmaceuticallyacceptable carriers and/or diluents.

The formulation of such pharmaceutical or veterinary compositions iswell known to persons skilled in this field. Suitable pharmaceuticallyacceptable carriers and/or diluents include any and all conventionalsolvents, dispersion media, fillers, solid carriers, aqueous solutions,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents, and the like. The use of such media and agents forpharmaceutically active substances is well known in the art, and it isdescribed, by way of example, in Remington's Pharmaceutical Sciences,18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofaras any conventional media or agent is incompatible with the activecomponent, use thereof in the compositions of the present invention iscontemplated. Supplementary active ingredients can also be incorporatedinto the compositions.

It is especially advantageous to formulate compositions in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform as used herein refers to physically discrete units suited asunitary dosages for the human subjects to be treated; each unitcontaining a predetermined quantity of active component calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier and/or diluent. The specifications for the noveldosage unit forms of the invention are dictated by and directlydependent on (a) the unique characteristics of the active component andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active componentfor the particular treatment.

A variety of administration routes are available. The particular modeselected will depend, of course, upon the particular condition beingtreated and the dosage that is determined to provide for optimaltherapeutic efficacy. The methods of this invention, generally speaking,may be practised using various modes of administration that aremedically acceptable, meaning any mode that allows for mucosal contactwith the active component of the invention without causing clinicallyunacceptable adverse effects. Mucosal administration, including theoral, nasal or intestinal routes, is preferred.

Optimal formulations for tolerogenic preparations of hybrid GAD 67/65 inaccordance with the present invention, for example simple aqueous orsalt solutions or other pharmaceutical compositions that are effectivewhen administered by a mucosal route, may be readily determined byroutine trial and experiment. Suitable formulations allow for thereestablishment of natural tolerance to the GAD molecule and therebyabrogate the harmful autoimmune reaction as discussed above.

Throughout this specification, unless the context requires otherwise,the word "comprise", or variations such as "comprises" or "comprising",will be understood to imply the inclusion of a stated integer or groupof integers but not the exclusion of any other integer or group ofintegers.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, a novel hybrid form of GAD hasbeen created, in one particular embodiment by expression of a nucleicacid sequence which is a fusion between the cDNA that encodes aminoacids 1-101 of the human GAD67 protein and the cDNA that encodes aminoacids 96-585 of the human GAD65 protein. This hybrid GAD67/65 has beenexpressed constitutively under the control of the highly activephosphoglycerate kinase promoter (PGKI) in the yeast S. cerevisiae.Thus, it has been demonstrated that substantial levels of anenzymatically active hybrid GAD can be readily produced in yeasts, suchas S. cerevisiae. Most significantly, this hybrid GAD could be purifiedby a single affinity chromatography step, and the purified hybrid GADhad the appropriate conformation to be highly reactive with sera ofpatients with IDDM, when such sera contain anti-GAD. The quantity ofpurified protein obtained in small scale preparations, 0.3-0.5 mg/liter,was ample for detailed studies at the molecular level. Larger quantitiesare readily obtainable by increasing the capacity of the fermentationvessels and increasing the capacity of the affinity column used, sincethere is no limitation in the supply of resources using the recombinantyeast. The ability to propagate yeast in large quantities at low cost isa great advantage over other mammalian expression systems. Beingeukaryotic, the yeast system is superior to the prokaryotic expressionsystems such as E. coli in terms of retention of post-translationalmodifications.

The N-terminal region of GAD65 is palmitoylated and hence is veryhydrophobic, and it is likely that this makes the purified polypeptidesstick to surfaces with which it comes in contact. This is a distinctdisadvantage in the preparation of recombinant GAD65. In the case ofhybrid GAD67/65, the N-terminal hydrophobic region of GAD65 is replacedby that of GAD67 which is more hydrophilic in nature. When this hybridGAD was synthesised by in vitro transcription and translation in rabbitreticulocyte lysate in the presence of ³⁵ S-methionine, the resultingproduct was shown to contain comparable reactivities to purified porcineGAD in radioimmunoprecipitation assays using IDDM sera. Therefore, theutility of expressing the hybrid GAD in yeast is three fold: it isenzymatically highly active, it is immunologically potent for IDDM sera,and it is far more readily recoverable during purification.

It will be appreciated that in addition to the affinity chromatographyprocedures described in detail herein, other appropriate proteinpurification procedures which are well known in the art may also be usedto produce a purified hybrid GAD67/65 product suitable for use indiagnostic immunoassays, or for use in treatment of a patient to inhibitor prevent IDDM by induced tolerogenesis as described above, forexample, in an orally delivered pharmaceutical composition.

Further features of the present invention are more fully described inthe following Examples. It is to be understood, however, that thisdetailed description is included solely for the purposes of exemplifyingthe present invention, and should not be understood in any way as arestriction on the broad description of the invention as set out above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Correlation of the reactivity of IDDM sera, as detected byimmunoprecipitation, between purified porcine brain GAD and RRLexpressed human brain GAD65. The correlation was significant (p<0.005) .

FIG. 2. Correlation of the reactivity of IDDM sera, as detected byimmunoprecipitation, between purified porcine brain GAD and RRLexpressed hybrid GAD67/65. The correlation was highly significant(p<0.001).

FIG. 3. Correlation of the reactivity of IDDM sera, detected byimmunoprecipitation, between RRL expressed human brain GAD65 and hybridGAD67/65. The correlation was significant (p=0.002).

FIG. 4 Autoradiograph of a SDS-PAGE gel of protein A Sepharose pelletsof IDDM and NHS after radioimmunoassay (RIA) with RRL expressed hybridGAD67/65 separated under (a) non-reducing and (b) reducing conditions.Pellets of IDDM sera highly reactive by RIA with hybrid GAD67/65 arecontained in lanes 1 and 2, weakly reactive with hybrid GAD67/65 in lane3, and non-reactive NHS in lanes 4-7. Under non-reducing conditions, 2bands of ˜130 and 110 kDa are precipitated by IDDM sera which, underreducing conditions, reduced to ˜65 and 45 kDa and smaller molecularweight bands. NHS did not precipitate either the 130 or 65 kDa band. Theparticular immunoreactivity of GAD with 130-110 kDa forms of themolecule under non-reducing conditions is described by Rowley et al.(1996).

FIG. 5 Schematic illustration of pMONBC6. The black regions represent E.coli DNA sequences, including sequences specifying ampicillin resistance(bla) and the bacterial plasmid replication origin (ori). The greyregions represent yeast DNA sequences, including the yeast plasmidreplication origin (2 μ), selectable marker LEU2 and transcriptionalcontrol sequences from the PGK1 gene, including the PGK1 promoter(PGK1p) and PGK1 terminator (PGK1t). The DNA sequence coding for hybridGAD67/65, in which amino acid residues 1-101 of GAD67 is fused to aminoacid residues 96-585 of GAD65 as illustrated, was cloned into theBamHI/NotI cloning site (shown as A) of the parent vector pAS-1 togenerate pMONBC6. Abbreviations: B, BamHI; H, HindIII; Bg, BglII; N,NotI.

FIG. 6 Analysis of hybrid GAD67/65 purified from yeast lysates. Yeastlysate collected from various stages of clarification duringpurification were studied as described (Methods). S1-4 are shown inlanes 1-3, 4-6, 7-9 and 10, respectively. F1-11 shown in lanes 11-21 arethe first 11 fractions (1 ml volume) collected during elution of hybridGAD from the GAD-1 affinity column. The yeast lysates studied wereprepared from YRD15 (lanes 1, 4 and 7), YpAS-1 (lanes 2, 5 and 8); andYGAD-2 (lanes 3, 6, 9 and 10). FIG. 6A is a Western Blot analysis usingGAD-6 and FIG. 6B is a silver staining analysis. 7.5% acrylamide wasused for the SDS-PAGE.

FIG. 7 A. Comparison of yeast hybrid GAD67/65 and purified porcine brainGAD by radio-immunoprecipitation. The sera tested were these submittedto the Second International GAD Workshop. Sera were analysed for thepresence of antibodies to GAD by radioimmunoprecipitation. The resultsare expressed as reference units (Rowley et al., 1992) according to astandard reference serum, defined as containing 100 units. Thecorrelation coefficient (r) between the recombinant yeast GAD andporcine brain GAD immunoprecipitation results is high, 0.94.

FIG. 7 B. Comparison of yeast hybrid GAD67/65 retained for 18 months at-20° C. and purified porcine brain GAD by radioimmunoprecipitation(RIP). The sera tested were a randomly derived collection from patientswith diabetes mellitus, and results are expressed as indicated in FIG.7A. The correlation coefficient (r) between the stored recombinant yeastGAD and porcine brain GAD immunoprecipitation results is high, 0.93.Comparability of results is optimal in the range of reliability of theRIP assay which is up to 100 units, beyond which titration of serum isrequired for precise quantification (Chen et al. 1993).

FIG. 8 Schematic illustration of pMONTP1. The coding for theconstruction of this vector, and abbreviations used, are as describedfor FIG. 5. Shown is the introduction at the 3' end of the cDNA forhybrid GAD67/65 of nucleotides encoding six histidine residues(hexahistidine tag)

EXAMPLE 1

This Example illustrates that hybrid GAD67/65 expressed by in vitrotranscription and translation using rabbit reticulocyte lysate is asfully effective as GAD65 in immunoassays to demonstrate anti-GADreactivity of serum in IDDM.

Materials and Methods GAD cDNA Clones

Brain-derived cDNAs encoding human brain GAD65 and brain GAD67, andfeline (F) brain GAD67 were gifts from Dr. A. Tobin. H-GAD65 and H-GAD67were both contained in the Bluescript-SK vector (Stratagene, USA),designated as pBSGAD65 and pBSGAD67 respectively, under the control ofthe T3 promoter. The 1755 bp coding region of GAD65 was contained in a2010 bp insert between the SacI and EcoRl cloning sites and the 1785 bpcoding region of GAD67 was contained in a 2700 bp insert in the EcoRIcloning site. The 1878 bp F-GAD67 was encoded between the EcoRl and XbaIcloning sites of the pGEM4 vector (Promega, USA), designated as pGEM-FGAD67, under the control of the SP6 promoter.

A hybrid molecule was constructed consisting of combined regions ofhuman brain GAD65 and GAD67, designated as GAD67(1-101)/GAD65(96-585)(hybrid GAD67/65), by replacing the region of cDNA encoding amino acids1-95 of GAD65 with the region of cDNA encoding amino acids 1-101 fromGAD67. The polymerase chain reaction (PCR) was used to amplifyselectively the DNA sequence encoding amino acids 1-101 of GAD67. Thesequence of the 5' oligonucleotide primer was (SEQ ID NO: 1) 5'TGGAGCTCATGGCGTCTTCGACCCCATCT 3' which incorporated a SacI restrictionsite at the 5' end. The sequence of the 3' oligonucleotide primer was(SEQ ID NO: 2) 5' TTCGCCGGCAGATCTCTAGCAAA 3' which incorporated a Bsr FIsite at the 3' end without altering any amino acid sequence encoded bythe H-GAD65 gene at the point of ligation. PCR using these 2 primerswith pBSGAD 67 resulted in a product of 303 bp. The GAD65 in theBluescript SK vector, pBSGAD 65, was prepared for the ligation of theGAD67 PCR fragment by first digesting with Sac I and then with Bsr FI.Due to the presence of multiple Bsr FI sites in the plasmid, ˜5 μg ofthe SacI digested GAD65 plasmid was subjected to time course digest with0.2 U of Bsr FI, aliquots were removed at 10, 20, 30, 40 and 60 minutesand examined by agarose gel electrophoresis. A 4.6 Kb DNA fragment ofthe digested product was recovered and ligated with the GAD67 PCRproduct. The resultant hybrid cDNA clone for GAD67/65₁ designated aspBSGAD67/65, was characterised by DNA sequence analysis. DNA sequencingof the fusion point between GAD65 and GAD67 in the resultant hybridclone confirmed that the recombinant molecule had been formed correctly.

Production of GAD

GAD was expressed by in vitro transcription and translation using therabbit reticulocyte lysate (RRL) system. Prior to transcription,plasmids pBSGAD65 and pBSGAD67/65 encoding human GAD65 and hybridGAD67/65 respectively, were linearized at the 3' non-coding region usingEcoRl and plasmid pGEM-F-GAD67 was linearized with Xba I. Transcriptionof 1 μg plasmid DNA was in a 20 μl reaction mixture containing 40 mMTris-HCl, pH 7.9, 6 mM MgCl₂, 2 mM spermidine, 10 mM dithiothreitol, 500μM of each of ATP, GTP, CTP and UTP, 100 μg/ml BSA, 1000 U/ml RNAsin(Promega) and 600 U/ml of either T3 or SP6 RNA polymerase. This mixturewas incubated for 60 minutes at 38° C. Transcription was stopped by theaddition of 5 μl of 5 mM EDTA to the transcription mixture. Atranslation cocktail consisting of 200 μl nuclease treated RRLsupplemented with 10 μl 1 mM amino acid mixture depleted of methionine,40 μl [³⁵ S-methionine (>1200 Ci/mmol) and 5 μl RNAsin (40,000 U/ml) wascombined before addition to the transcription mixture. The translationreaction was incubated for 30 minutes at 30° C. After incubation, themixture was centrifuged and the supernatant stored at -80° C. as 50 μlaliquots. Prior to use in the radioimmunoprecipitation assay, RRLtranslated GAD was passed through a NAP5 column (a Seogadex G-25 columnmade by Pharmacia Biotech) to separate the translation products fromfree [³⁵ S]-methionine.

Sera

The sera studied were from 50 patients with IDDM diagnosed according tothe criteria of the National Diabetes Data Group (1979). These sera werepre-selected according to positivity for autoantibodies to porcine brainGAD according to a radioimmunoprecipitation assay (see below). Controlswere derived from a pool of sera from 50 healthy subjects.

Radioimmunoprecipitation Assay for anti-GAD Using Porcine Brain GAD

GAD was prepared from porcine brain by differential centrifugation andpassage of the supernatant through an affinity column conjugated withthe monoclonal antibody, GAD1. The ensuing preparation containing amixture of GAD65 and GAD67 was labelled with ¹²⁵ I using the chloramineT procedure. These methods are described by Rowley et al, 1992.

Radioimmunoprecipitation Assay Using GAD Produced in RRL

Gel-purified GAD preparations obtained using RRL were "pre-cleared" withnormal human sera (NHS) prior to assay. Two hundred μl of NHS was addedto 200 μl of purified GAD and held for 60 minutes at 4° C. Two hundredμL of 50% Protein A Sepharose (Pharmacia, Sweden) suspended in washbuffer 120 mM Tris, 150 mM NaCl, 0.5% (w/v) Triton X-100, pH 7.4] wasthen added and held with the NHS and rabbit reticulocytelysate-expressed GAD for 60 minutes at 4° C. The mixture was centrifugedfor 2 minutes at 690×g and the supernatant was used as the source ofantigen. Immunoprecipitation was performed by adding 40,000 DPM of"pre-cleared" GAD, 25 μl test sera and wash buffer in a total volume of50 μl . After a 16 hr incubation at 4° C., 50 μl of 50% Protein ASepharose was added and incubated for 60 minutes at 4° C. 1 ml of washbuffer was added to each of the samples, the samples were centrifugedfor 2 minutes at 690×g, to pellet the Protein A Sepharose beads, and thesupernatant was discarded. The pellets were washed an additional 3times, then vortexed vigorously with 1 ml of scintillant and counted ina gamma counter.

Immunoprecipitated Form of Hybrid GAD67/65

The molecular weight of the hybrid GAD67/65 that was precipitated byIDDM sera was examined. After immunoprecipitation, the protein ASepharose pellets were separated by SDS-PAGE on 10% gels under reducingand non-reducing conditions. The gels were fixed for 60 minutes in 50%(v/v) methanol, 10% (v/v) acetic acid, and then for 30 minutes in 5%(v/v) methanol, 10% (v/v) acetic acid, and immersed in Amplify solution(Amersham, UK) for 60 minutes before being dried onto filter paper undervacuum. The gels were overlayed with Fuji Medical X-Ray film (Fuji,Australia) in a cassette for 2 weeks at -80° C. before being developed.

Statistical Analysis

Data were analysed using the Complete Statistical System (StatSoft Inc,USA) computer program. Data were calculated as arithmetic means andstandard deviations. The significance of differences was tested byStudent's t-test for independent samples. Correlations were sought usingthe Pearson product moment correlation coefficient test.

Results Reactivity of IDDM Sera with GAD of Differing Provenance

The frequency of reactivity by RIA of NHS and IDDM sera with recombinantGAD65 recombinant F-GAD67 and hybrid GAD67/65, all expressed in therabbit reticulocyte lysate system, was determined and is shown inTable 1. The cut-off for a positive reaction for each antigen wasdefined as 3 standard deviations (SD) above the mean DPM precipitated bythe NHS (Table 1). It is to be noted that these sera were pre-selectedfor reactivity by RIA with GAD purified from porcine brain. Thefrequency of positive reactions was 90%, 40% and 92% for GAD65, F-GAD67and hybrid GAD67/65 respectively. Of the 40% of IDDM sera that reactedwith F-GAD67, all reacted also with GAD65 and hybrid GAD67/65. Of 4 IDDMsera that did not react with hybrid GAD67/65, 2 did react with GAD65 butnot F-GAD67, and 2 were entirely non-reactive.

                  TABLE 1                                                         ______________________________________                                        Results.sup.1 of immunoprecipitation assays with normal human sera (NHS)       and insulin-dependent diabetes mellitus sera (IDDM) using various             sources of recombinant GAD expressed in the rabbit reticulocyte lysate        system.                                                                                                                  Positive.sup.2                                                                   NHS Cut-off  IDDM IDDM:                                                     GAD n.sup.3 mean (SD) x +                                                    3SD n.sup.3 mean (SD)             ______________________________________                                                                                    NHS                               GAD65  48    156 (55)  321    50  1077 (444)                                                                            90:0                                  F-GAD67 45 182 (152) 638 50  871 (794) 40:2                                   Hybrid 50 572 (107) 893 50 4295 (1812) 92:0                                   GAD67/                                                                        65.sup.4                                                                    ______________________________________                                         .sup.1 Expressed as DPM in precipitate.                                       .sup.2 Positive = % of cases > mean + 3SD for NHS.                            .sup.3 Number of cases tested.                                                .sup.4 GAD67(1-101)/GAD65(96-585).                                       

Correlations were sought for between reactivities of IDDM sera withpreparations of GAD of differing provenance. Significant correlationswere found for the reactivity of IDDM sera with purified porcine brainGAD and recombinant H-GAD65 (p<0.001, R=0.670)--see FIG. 1, betweenpurified porcine brain GAD and hybrid GAD67/65 (p<0.001, R=0.6722)--seeFIG. 2, and between recombinant H-GAD65 and hybrid GAD67/65 (p<0.002,R=0.675)--see FIG. 3. No correlations were found between reactivities ofIDDM sera with porcine brain GAD and recombinant F-GAD67, recombinantGAD65 and recombinant F-GAD67, or hybrid GAD67/65 and recombinantF-GAD67.

Form of Hybrid GAD67/65 Precipitated by IDDM Sera

Hybrid GAD67/65 that was reactive with the sera of IDDM and normalsubjects was examined by SDS-PAGE under reducing and non-reducingconditions and autoradiography. Protein-A Sepharose pellets obtainedafter immunoprecipitation with ³⁵ S-labelled hybrid GAD67/65 from 3 IDDMpatients and 4 normal human subjects showed that under non-reducingconditions, IDDM sera precipitated 2 bands of ˜130 and 110 kDa and underreducing conditions, in the presence of β-mercaptoethanol, these bandsreduced to ˜65 and 45 kDa (FIG. 4).

EXAMPLE 2

This Example shows the derivation of vectors for the expression ofhybrid GAD67/65 in yeast, the purification of the expressed product byantibody affinity procedures, and the use of the radiolabelled productin radioimmunoprecipitation assays.

Materials and Methods Microbial Strains and Vectors

Escherichia coli strain DH5α [recAI endA1 gyrA96 thi-l relA1 SupE44ΔlactU169 (φ80 lacZΔM15) hsdR17] was used for all sub-cloning purposes.Saccharomyces cerevisiae strain YRD15 (MATa ura3-2513-373, his3-11,leu2-3-l1), a generous gift from Dr R. J. Devenish (Department ofBiochemistry and Molecular Biology, Monash University, Clayton,Australia), was used for expression and purification of the humanrecombinant GAD. For most of the yeast expression experiments aconstruct based on plasmid pAS-1 was employed (see below for details),but in early studies a construct based on pYES2 (Invitrogen) wasemployed.

The yeast expression vector pAS- 1, a derivative of pYEPLAC191 (Gietzand Sugino 1988), was obtained from Dr A. Stratton and Dr. R. J.Devenish (Department of Biochemistry and Molecular Biology, MonashUniversity, Clayton, Australia). It contains the yeast phosphoglyceratekinase (PGK1) promoter and terminator elements to drive a high-levelconstitutive expression of heterologous genes, a 2 μm origin ofreplication to provide high copy number, and a LEU2 selectable marker tomaintain the plasmid in the host strain (refer to FIG. 5).

A plasmid pMALGAL67/65 was derived from pBSGAD67/65 (see Example 1) bydigestion of the plasmid pMALGAD67, containing the H-GAD67 cDNA in theEcoRl site of pMAL, with SalI and partially with NcoI. A 6243 bpfragment was purified and ligated to a 1876 bp fragment liberated bydigestion of pBSGAD67/65 with SalI and NcoI. Thus, plasmid pMALGAD 67/65was derived with a DNA sequence coding for maltose binding protein (MBP)fused to amino acids 1-101 of H-GAD67 which in turn are fused to aminoacids 96-585 of H-GAD65 (hybrid GAD67/65).

Construction of pMONBC6, the Yeast Expression Vector pAS-1 Bearing theHybrid GAD67/65.

The construction of vectors for expression of hybrid GAD in yeast wasaccomplished in two stages. In the first stage, the hybrid GAD moleculewas sub-cloned from pMALGAD67/65 into the EcoRI site of the induciblepromoter system pYES2 to make pMONBC3. This plasmid, which carries theGALl promoter, was transformed into yeast YRD15, but the yield ofrecombinant hybrid GAD was low (data not shown). Hence, the plasmidpAS-1 containing the PGK1 promoter which provides a constitutiveexpression was tested. The BamHI-NotI fragment bearing the hybridGAD67/65 gene was re-isolated from pMONBC3, and subcloned into theBglII-Notl site of pAS-1, to generate the plasmid pMONBC6 (FIG. 5).

Expression of Recombinant Hybrid GAD67/65 in S. cerevisiae.

The YRD15 strain was transformed with the plasmid pMONBC6 using alithium acetate method (Elble, 1992). Transformants (YGAD-2) wereselected on synthetic minimal glucose medium, SD+ura+his comprising0.67% yeast nitrogen base without amino acid, 2% glucose, 20 mg/L uraciland 20 mg/L histidine. For expression, overnight cultures were grown upat 28° C. in liquid SD+ura+his medium with shaking, then diluted 1:50into a selective medium (sacc) comprising 1 % yeast extract, 10%glucose, 20 mg/L uracil, 20 mg/L histidine, 0.12% (NH₄)₂ SO₄, 0.1% KH₂PO₄, 0.07% MgCl₂, 0.05% NaCl, 0.01% CaCl₂ and 0.005% FeCi₃ and grown fora further 18-20 hours at 28° C. with shaking. The high glucoseconcentration was chosen to favour maximal induction of the PGK1promoter. After growth, the cultures were harvested by centrifugation at4° C. and used for enzyme purification. As a control, YRD15 was alsotransformed with the parent plasmid pAS-1 as above, and the resultingtransformants (YpAS-1) as well as the yeast host YRD15 were used asnegative controls in the studies of hybrid GAD expression in YGAD-2.

Purification of Recombinant Hybrid GAD67/65 from S. cerevisiae

A crude lysate of the yeast cells was obtained by vortexing the cellswith equal volumes of glass beads (425-600 μm diameter) and lysis buffer(50 mM KH₂ PO₄, 1 mM EDTA, 1 mM aminoethylisothiouronium bromide (AET),20 μM pyridoxal phosphate (PLP), 10 mM 2-mercaptoethanol, 0.5 mMphenylmethylsulfonyl fluoride (PMSF) and 10 mM glutamate, pH 7.2), insix 30 second pulses, with 30 seconds on ice between pulses. The lysatewas separated from the beads and intact cells by centrifugation, 5000 gfor 5 min. The beads and the cell pellet were resuspended in anothervolume of lysis buffer, vortexed and centrifuged as above, and thelysate collected was combined with the first supernatant. The pooledlysate (S1) was clarified by centrifugation at 18,500×g for 10 min togenerate S2 which was further clarified by ultra centrifugation,100,000×g for 60 min. The supernatant collected was filtered through a0.2 μm filter to remove any floating materials, to generate S3.Purification of recombinant hybrid GAD in S3 was by affinitychromatography using the monoclonal antibody GAD-1 (Chang and Gottlieb,1988) conjugated to cyanogen bromide activated-Sepharose, as forpurification of porcine brain GAD (Rowley et al., 1992). Recombinanthybrid GAD bound to the column was eluted into 1 ml fractions (F) with ahigh pH buffer (pH 10.5), and these were immediately neutralized to pH7. A portion of the S1-3 and the flow through (S4) as well as all theeluted fractions were stored at -20° C. in 30% glycerol prior to theiruse in protein estimations and enzyme assays, other analytical studiesincluding SDS-PAGE, immunoblotting, and iodination forimmunoprecipitation.

Characterisation of Fractions Collected Throughout GAD PurificationProcess

Lysates (S1-S4) and purified fractions (F1-11 ) were analysed for thepresence of recombinant hybrid GAD by SDS-PAGE and immunoblotting usingthe monoclonal antibody GAD-6 (Chang and Gottlieb, 1988) which reactswith the GAD65 isoform. The epitope for GAD-6 has been mapped to theC-terminal region of GAD65 (Daw and Powers, 1995); thus it was expectedthat GAD-6 would react with hybrid GAD67/65. GAD enzymatic activity wasdetermined by a spin column chromatography procedure that uses the anionexchange resin (Bio-Rad AG® 1 -X8) to separate ³ H-GABA from the morehighly acidic ³ H-glutamate, as described previously (Rowley et al.,1992). Protein concentration was determined by the Bradford dye-bindingprocedure (Bio-Rad Protein Assay) in accordance with the manufacturer'sinstructions.

Immunoprecipitation of Iodinated Recombinant Hybrid GAD67/65 by IDDMSera

Immunoprecipitation of iodinated recombinant hybrid GAD67/65 by IDDM andnon-IDDM sera was performed as described previously (Rowley et al.,1992). The 100 sera analysed were those submitted to the SecondInternational GAD Antibody Workshop (Paris, France, 1994). The resultsobtained using the radio-iodinated yeast hybrid GAD67/65 were comparedwith those obtained using the standard preparation of porcine brain GADfrom the laboratory (Rowley et al., 1992; Chen et al., 1993).

Results Expression of Recombinant Hybrid GAD67/65 in YGAD-2

The expression of hybrid GAD67/65 in recombinant yeast YGAD-2 wasstudied by enzyme assay and Western blot on the crude cell lysate S1.Appreciable amounts of GAD protein and degrees of GAD enzyme activitywere detectable. The results of the GAD enzyme activity in S1 preparedfrom the recombinant yeast YGAD-2, as well as all the controls YRD15 andYpAS-1 are shown in Table 2 . A high level of GAD enzyme activity wasobserved from the recombinant yeast, 8.4 nmol/min/mg, significantlyhigher than that from the yeast host YRD15, 0.68 nmol/min/mg, and thecontrol YpAS-1, 0.73 nmol/min/mg. This level of enzyme activity is wellabove the levels reported previously for GAD in brain homogenates thathave been widely used for GAD preparations; for example, that for humanand pig brain homogenates was reported to be 0.124 and 2.4 nmol/min/mg,respectively (Blindermann et al., 1978; Spink et al., 1985), althoughthe GAD enzyme activity in these brain homogenates might well varysignificantly according to the freshness of the materials. The highlevel of GAD enzyme activity in the yeast lysates, as well as theability to control the quality of the yeast material, are clearadvantages of the GAD purification process from yeast.

                                      TABLE 2                                     __________________________________________________________________________    Purification of recombinant hybrid GAD expressed in Saccharomyces             cerevisiae*.                                                                       Protein      Specific GAD enzyme                                                                     Total GAD                                            concentration Total Protein activity enzyme activity  Purification                                                   Sample (                                                                     μg/ml) (mg) (mmol/min/mg)                                                  (nmol/min) % Recovery (fold)         __________________________________________________________________________    YGAD-2                                                                          S1 5155 232 8.4 1949 100  1                                                   S2 4688 210 7.5 1579 81 --                                                    S3 4007 180 4.8 864 44.4 --                                                   S4 4176 188 0.6 113 5.8 --                                                    F1 0  0 -- -- -- --                                                           F2 2.34 3.5 × 10.sup.-3 0 -- -- --                                      F3 3.83 5.7 × 10.sup.-3 0 -- -- --                                      F4 5.6 8.4 × 10.sup.-3 0 -- -- --                                       F5 33.83  51 × 10.sup.-3 2660 135 -- --                                 F6 63.58  95 × 10.sup.-3 2916 277 -- --                                 F7 42.47  63 × 10.sup.-3 2041 129 -- --                                 F8 22.85 34.3 × 10.sup.-3   829 28.4 -- --                              F9 7.1 10.7 × 10.sup.-3   1021 10.9 -- --                               F10 9.18 13.8 × 10.sup.-3   454 6.3 -- --                               F11 9.47 14.2 × 10.sup.-3   212 3 -- --                                 F (5-10).sup.1 29.84 0.27 1653 2.446 22.9 197                                 YRD-15                                                                        S1 4089 184 0.68 -- -- --                                                     S2 3760 169 0.6 -- -- --                                                      S3 3740 168 0.65 -- -- --                                                     YpAS-1                                                                        S1 4076 212 0.73 -- -- --                                                     S2 4397 198 0.59 -- -- --                                                     S3 3823 172 0.85 -- -- --                                                   __________________________________________________________________________     *10 ml harvested packed cells were lysed in the presence of glass beads.      S1-S4 were generated as described in the methods, where S1 was collected      after centrifugation at 5,000 g for 5 min to remove intact cells and glas     beads; S2 was collected after the lysate was further clarified by             centrifugation at 18,500 × g for 10 min, S3 was collected after         ultracentrifugations at 100,000 × g for 2 × 60 min and            filtration through a 0.2 μm membrane to remove floating material;  # S     was assigned to the flowthrough from the column chromatography using GAD1     Recombinant hybrid GAD bound to the affinity column containing GAD1 was       eluted in 1 ml fractions (F1-11) of which the analysis of these 11            fractions (F1-11) is shown.                                                   .sup.1 F5-10 is a pool of F5 to F10.                                     

Using monoclonal antibody GAD-6 directed to GAD65 on Western blots hasrevealed at least two major GAD products in the yeast lysate fromYGAD-2. One of those corresponded to 64 kDa, and the other to 60 kDa(FIG. 6A, lane 3). The 64 kDa product corresponded well to the fulllength of the hybrid GAD67/65 construct, whilst the 60 kDa product wouldrepresent an N-terminally truncated hybrid GAD product (see below).Based on the relative intensity of the two bands, the amount of the falllength product would be around 2-3 fold greater than that of thetruncated product. A minor product corresponding to 45 kDa was alsoobserved but its identity was not further investigated. Western blotanalysis on cell lysates prepared from strains YRD15 and YpAS-1 showedno signal (FIG. 6A, lanes 1 and 2). Similarly, no signal was detectableon the culture medium in which YGAD-2 was propagated (data not shown).

These results of GAD enzyme activity and Western blot on S1 suggestedthat the recombinant hybrid GAD67/65 was expressed at a high level andwith very high enzyme activity and, seemingly, was stable in the cytosolof the yeast host.

Purification of Recombinant Hybrid GAD67/65

Although there was an initial loss of GAD protein during its preparationfrom yeast lysate, a highly enzymatically reactive and pure preparationof GAD could be obtained by a single-step affinity chromatography, usingthe monoclonal antibody GAD-1. The procedure for purification ofrecombinant hybrid GAD from yeast was as described for porcine brain(Rowley et al., 1992). The amount of GAD recovered from various stages,S1-S4 and F1-F11, of the preparation of hybrid GAD was monitored by GADenzyme assays, SDS-PAGE and Western blots. The results from a typicalpreparation are shown in Table 2 and FIG. 6A. Considering the enzymeactivities (Table 2), there was a slight decrease of enzyme activityfrom 8.4 to 7.5 nmol/min/mg for S1 and S2, respectively. However for S3,there was a substantial decrease of the enzyme activity to 4.8nmol/min/mg after the ultracentrifugation at 100,000 g. This loss ofhybrid GAD during the ultra-centrifugation was regarded as a compromisefor clarity of the lysate, which was essential for the columnchromatography procedure. Nevertheless, this amount of GAD activity thatremained in S3 is still twice as much as that reported for preparationsfrom porcine brain, 2.4 nmol/min/mg (Spink et al., 1985)

As shown in Table 2, upon incubation with the GAD-1 antibody on thecolumn, the enzyme activity became negligible in the flow through S4,0.6 nmol/min/mg, showing that the recombinant yeast hybrid GAD boundefficiently to the GAD-1 antibody in the column. The immobilisedrecombinant hybrid GAD was eluted off the column with a pH 10.5 buffer,1 ml fractions were collected and 0.5 g of glycerol was added to eachfraction, as for S1-S4. The samples were kept at -20° C. beforesubsequent analyses were carried out. The results of analyses of thefirst 11 fractions (F1-11) collected were as follows. The purified yeastrecombinant hybrid GAD was enzymatically highly reactive (Table 2),since the specific GAD enzyme activity was up to 2,916 nmol/min/mg inthe peak fraction (F6). Pooling F5-10 gave a total yield of purifiedhybrid GAD of 270 μg with an average enzyme activity of 1,653nmol/min/mg. The pooled F5-10 fractions, regarded as the net yield,represented a purification of 197 fold with a recovery of 22.9% ofhybrid GAD in S1. Although the amount of hybrid GAD purified per runcould be further improved, such as by increasing the capacity of theaffinity column used, this enzyme activity, 1,653 nmollmin/mg, is veryhigh compared with that of GAD purified from human brain which is 1,000nmol/min/mg (Blindermann et al., 1978).

Studies on the control samples (S1-3) prepared from the yeast host(YRD15) and the yeast host transformed with the plasmid without theinsert (YpAS-1) revealed negligible amounts of GAD enzyme activity(Table 2).

On Western blot analysis using the monoclonal antibody GAD-6, the amountof detectable hybrid GAD67/65 in S3 had decreased significantly comparedwith that of S1 and S2 (FIG. 6A, lanes 9, 3 and 6, respectively). Thisagrees well with results of the GAD enzyme activity assays, and suggeststhat ultra-centrifugation led to a significant loss of hybrid GAD fromthe lysate. It should be noted that the ratio of the 64 kDa to the 60kDa proteins remained unchanged in samples S1-3. FIG. 6A also shows theresults of Western blot analysis of the purified hybrid GAD (F1-F11,lanes 11-21). Recombinant hybrid GAD is detectable in F5-F10 (lanes16-20), with F6 containing the highest level of hybrid GAD detectable(lane 17). This is in agreement with results of the specific enzymeactivity assays (Table 2 ). The hybrid GAD products detectable representas least two sizes, 64 and 60 kDa, as mentioned above. Interestingly,compared with the yeast lysates, the 60 kDa products in the purifiedsamples had increased 34 fold over the 64 kDa product (FIG. 6A). The 40kDa product observed previously was barely detectable.

The results of silver staining of SDS-PAGE analyses are shown in FIG.6B. The hybrid GAD preparation is very pure (lanes 16-20), and F6 hasthe highest content of protein products. However, there are at least 5distinct bands detectable, corresponding to molecular sizes, 64, 60,59.5, 59 and 58.5 kDa. Previous studies in this laboratory havedemonstrated that porcine GAD preparations are prone to N-terminaltruncations, generating products from 65 to 55 kDa (Tuomi et al., 1994).Therefore it was expected that the yeast recombinant hybrid GAD was alsotruncated to yield a range of products of slightly lower molecularweight but with retained immunoreactivity. The Western blot analysis(FIG. 6A lanes 15-20) further suggests that truncation of the fulllength hybrid GAD occurs at the N terminal region of the protein, inthat the GAD-6 monoclonal antibody used recognizes the C-terminal regionof the GAD molecule, amino acids 529-586 (see above). It would beexpected that all of the four truncated products, ranging in size from60-58.5 kDa, would retain the GAD6 recognition domain, but this has yetto be ascertained since Western blot analysis using enhancedchemiluminescence (ECL) does not allow resolution of these fourcomponents into distinct bands. Importantly, either part or all of thesepurified hybrid GAD components are enzymatically highly reactive, andimmunoprecipitable by IDDM sera (see below).

It would be desirable to prevent or limit the proteolysis that occursduring purification, by including protease inhibitors in the process.Two experiments thus far have been carried out to investigate whetherthe above mentioned lower molecular weight N-terminal truncationproducts of hybrid GAD67/65 can be limited by protease inhibitors. Inthe first experiment, a protease inhibitor cocktail (1 mg/ml PefablocSC, 3 μg/ml Leupeptin and 15 μg/ml Pepstatin) was included in the lysisbuffer above; in the second, the composition of the cocktail wasslightly different (25 μg/ml Aprotinin, 1 μ/ml Leupeptin and 2.5 μg/mlPepstatin A). These were ineffective in that the resulting GAD was foundnot to differ from untreated GAD with regard to enzyme activity,immunoreactivity on Western blot, or protein content as judged bySDS-PAGE. The next step would be determination of the specific type ofproteases involved, for example by N-terminal sequencing of thesetruncated products.

Characterisation of Purified Recombinant Hybrid GAD67/65 by Reactivityto IDDM Sera

The reactivity of the yeast hybrid GAD67/65 with IDDM sera was assayedby radioimmunoprecipitation assay using ¹²⁵ -labelled protein with apanel of 100 sera derived from the Second International GAD workshop(Paris, France, 1994). These sera had previously been tested in thislaboratory using a preparation of GAD derived from porcine brain. Asshown in FIG. 7A, there was very good agreement between the resultsobtained using yeast hybrid GAD67/65 and porcine brain GAD. All 55 seraidentified as positive for anti-GAD using porcine brain GAD were alsoreactive to yeast hybrid GAD67/65, and all 45 sera identified negativefor anti-GAD using porcine brain GAD were non-reactive using the yeasthybrid GAD. Thus, the yeast hybrid GAD67/65 and the porcine GAD whenused in the standard radioimmunoprecipitation assay in this laboratoryexhibited identical sensitivity and specificity for the diagnosis ofIDDM.

Field Testing of Recombinant Hybrid Yeast GAD67/65

A preparation of recombinant yeast hybrid GAD67/65 has been"field-tested" as the standard reactant in radioimmunoassays fordetection of anti-GAD in sera from control subjects, and in individualswith various types of diabetes mellitus and other diseases. In total,5,068 individual serum assays have been performed in batches. Theserepresent assays for purposes of research publications and medicaldiagnosis and have not been "controlled" by a comparison with an assayusing an alternative form of GAD. Included in each assay batch werecontrol sera that are known to react as low positive units for anti-GAD,and (c) high positive units for anti-GAD. Throughout the test period,the assay runs have given a true performance as judged by consistency ofresults with the control sera and by absence of anomalous results withthe 5,068 test sera.

EXAMPLE 3

This Example illustrates that recombinant hybrid GAD67/65 produced in ayeast expression system retains stability when used as an immunoassaysubstrate (a) after labelling with radioactive iodine and (b) afterretention in storage for periods up to two years at -20° C. Two of thepurified samples with the highest content of GAD, from a yeast GADpreparation and fractionation as described in Example 2, were stored fortwo years at -20° C., radioiodine labelled and then compared forimmunoreactivity by radioimmunoassay using a standard laboratorypreparation of radiolabelled porcine brain GAD.

Materials and Methods

The sera used comprises 164 samples derived from normal subjects andpatients with diabetes mellitus for which there was a known range oflevels of anti-GAD, as judged by assays using radiolabelled purifiedporcine brain GAD.

The fractions used to assess effects of storage were those designated asF8 and F9 from a fractionation procedure performed as described inExample 2. These fractions were those most highly enriched for hybridGAD67/65. The fractions were stored at -20° C. in 30% w/v glycerol for 2years. Glycerol is added to prevent freezing and consequent degradationof GAD after thawing prior to use.

The fractions retrieved from storage were thawed and brought to roomtemperature and labelled with radioactive iodine. At the same time, apurified fraction from a freshly prepared hybrid GAD67/65 was alsolabelled with radioactive iodine.

Results

Two sets of comparisons are shown for three preparations of hybridGAD67/65. The first comparison in Table 3 shows results using (1) thestandard laboratory reference serum with a designated level ofreactivity of 100 units, (2) a control "high positive" serum, (3) acontrol "low positive" serum, and (4) pooled normal human sera. Thistable shows counts of radioactivity precipitated, and the derived unitsof anti-GAD activity, for a preparation of freshly derived hybrid yeastGAD67/65, a preparation of hybrid yeast GAD67/65 stored at -20° C., anda fresh preparation of hybrid yeast GAD with a polyhistidine tag (seeExample 4). The results are highly comparable for the threepreparations, indicating that neither long storage at -20° C. nor thegenetically engineered introduction of a polyhistidine tag (see below)has adverse effects on the immunoreactivity of yeast GAD.

                  TABLE 3                                                         ______________________________________                                        Comparison of antigenic reactivity of various preparations of                   recombinant hybrid yeast GAD67/65.                                                      Preparation of Autoantigenic GAD.sup.1                                                              "Tagged"                                       "Fresh" Yeast "Stored" Yeast Yeast                                            GAD67/65.sup.2 GAD67/65.sup.2 GAD67/65-H6.sup.4                            Serum Sample                                                                              cpm.sup.5                                                                            units   cpm  units cpm   units                             ______________________________________                                        Anti-GAD reference                                                                        8824   100     8604 100   13934 100                                 serum designated as                                                           100 units                                                                     High positive anti- 8975 102 8654 100.5 14497 104                             GAD serum                                                                     Low positive anti- 2198 25 2878 33  4606 33                                   GAD serum                                                                     Pooled normal human  250 3.0  177 2.0  606 3.6                                sera                                                                        ______________________________________                                         .sup.1 Various preparations of radioiodine labelled yeast GAD were            compared for reactivity with standard serum samples used for calibration      of routine RIP assays for antiGAD in this laboratory. These include the       standard reference serum designated as containing 100 units of antiGAD        activity, a known highpositive serum, a known lowpositive serum, and          pooled normal human sera.                                                     .sup.2 "Fresh" yeast GAD67/65 was iodinated within 4 weeks of preparation     specific activity 25 μCi/μg.                                            .sup.3 "Stored" yeast GAD67/65 was prepared and stored in 30% glycerol at     -20° C. for 18 months prior to testing, specific activity on           iodination was 22.6 μCi/μg.                                             .sup.4 "Tagged" yeast GAD67/65 was a fresh preparation with a                 hexahistidine tag (see FIG. 8).                                               .sup.5 cpm = counts of radioactivity precipitated in the radioimmunoassay

A comparison was made, using 164 sera from patients with diabetesmellitus with varying levels of anti-GAD, between radioimmunoassayresults for purified porcine brain GAD and the yeast GAD preparationthat had been retrieved from storage and labelled with radioactiveiodine. These results are shown in FIG. 7B as a scatterplot. The closeconcordance of results is indicated by the correlation coefficient of0.93. The correlation between immunoassay results for radioiodinelabelled yeast GAD67/65 and porcine brain GAD is in fact higher than thecorrelation shown in FIG. 2 between biosynthetically labelled (³⁵ S)yeast GAD and porcine brain GAD. It is evident from FIG. 7B that thereis a divergence from a linear correlation plot for the highest levels ofanti-GAD. This is in line with a known characteristic of the assay (Chenet al., 1993) for which, when there is antibody excess, titration ofserum is required for a meaningful quantitative result.

EXAMPLE 4

This Example illustrates that hybrid GAD67/65 can be endowed with aC-terminal hexahistidine (H6) tag so that if desired the expressed geneproduct can be purified through metal matrix columns with affinity forthe H6 tag, and that GAD67/65-H6 is enzymatically and antigenicallyequivalent to hybrid GAD67/65 purified by antibody-affinity columns.

Materials and Methods Construction of GAD67/65-H6

A second hybrid molecule was constructed using the original hybridGAD67/65 contained in the Bluescript-SK vector (Stratagene, USA),designated GAD67/65-H6. The polymerase chain reaction (PCR) was used toendow a C-terminal hexahistidine tag sequence on hybrid GAD67/65. Thesequence of the 5' oligonucleotide primer was (SEQ ID NO: 3) 5'CCGGAATTCACCATGGCGTCTTCGACCCC3', which incorporated an EcoRI site at the5' end but did not alter the amino acid sequence of GAD67/65. Thesequence of the 3' primer was (SEQ ID NO: 4) 5 'CCGGAATTCTTAGTGGTGGTGGTGGTGGTGAGATAAATCTTGTTCAA GGCGTTCTATTTCTTC3',which also incorporated an EcoRI site at the 3' end after the sequenceencoding the hexahistidine (H6) tag. A 1.8 kb DNA fragment encodingGAD67/65-H6 was recovered and digested with EcoRI, and sub-cloned intothe Bluescript vector above. DNA sequencing of the resultant cloneconfirmed that the recombinant molecule had been formed correctly.

Construction of pMONTP1, the Yeast Expression Vector Bearing GAD67/65-H6

The yeast expression vector pAS-1 was initially exposed to digestionwith BglII, followed by generation of "blunt ends" by use of the Klenowfragment of DNA polymerase I and then digestion with the NotIrestriction enzyme. The GAD67/65-H6 gene was isolated from theBluescript vector as an EcoRV-NotI fragment, and subcloned into pAS-1,to generate the plasmid pMONTP1, as illustrated in FIG. 8.

Expression of GAD67/65-H6 in S. cerevisiae.

The YRD-15 strain was transformed with the plasmid pMONTP1 using thelithium acetate method (Elble, 1992). Transformants (YGAD-3) wereselected and expressed for GAD production as for YGAD-2, which is theYRD-15 strain transformed with plasmid pMONBC6 and expressing theoriginal recombinant hybrid GAD67/65.

Purification of GAD67/65-H6from S. cerevisiae.

Purification of recombinant GAD from YGAD-3 was performed as for YGAD-2,except for a differing composition of the lysis buffer: this was 50 mMKH₂ PO₄, 1 mM EDTA, 1 mM aminoethylisothiouronium bromide AET, 20 μMpyridoxal-L-phosphate PLP, 10 mM 2-mercaptoethanol, 10 mM glutamate,0.12% (v/v) Triton X-100 and 0.5 mM phenylmethylsulfonyl fluoride(PMSF), pH 7.2.

Results

Expression of recombinant hybrid GAD67/65-H6 in YGAD-3.

The expression of GAD67/65-H6 in recombinant yeast YGAD-3 was performedin a manner very similar to that for GAD67/65 from YGAD-2. The resultsof the GAD enzyme assay for the crude yeast cell lysates did not revealsignificant differences, these being 4.4 nmol/min/mg for GAD67/65 versus3.6 nmol/min/mg for GAD67/65-H6. These were substantially higher thanthe enzyme assay results for a lysate of yeast host YRD-15, 0.68nmol/min/mg, and the control yeast YpAS-1, 0.73 nmol/min/mg. Using themonoclonal antibody GAD-6 reactive with the C-terminal moiety on GAD65by Western blot, two major GAD products in the yeast lysate from YGAD-3are seen, with molecular weights of 64 and 60 kDa respectively, as forYGAD-2. After passage through the GAD-1 monoclonal antibody affinitycolumn, the GAD67/65-H6 was purified as for GAD67/65. The yields were0.18 mg of GAD67/65-H6 which compares well with 0.28 mg of GAD67/65,from equivalent 500 ml cultures of yeast. There was slightly higherenzyme activity in the pooled fractions for GAD67/65-H6 (4,654nmol/min/mg). Purification levels were also very similar, 197-fold forGAD67/65 versus 239-fold for GAD67/65-H6.

Reactivity by Radioimmunoprecipitation After Labelling with RadioactiveIodine

The introduction of a polyhistidine (H6) tag was shown not to influencethe reactivity of recombinant hybrid yeast GAD67/65. Table 3 above showsresults by radioimmunoprecipitation in counts of radioactivityprecipitated, and conversion to assay units, for a fresh preparation ofhybrid GAD67/65, a stored preparation of hybrid GAD67/65, and apreparation of hybrid GAD67/65-H6 after purification on an antibodyaffinity column. Data are presented for the standard reference serumdesignated as having 100 units of activity, a high positive anti-GADserum, a low positive anti-GAD serum, and pooled normal human sera. Itis seen that results are comparable for the three preparations.

EXAMPLE 5

This Example describes a preparation of recombinant hybrid yeastGAD67/65 that is purified and lyophilized in 1-2 mg quantities suitablefor reconstitution in a vehicle in which GAD can be delivered to theupper intestinal tract of a strain of mice (non-obese diabetic, NOD,mice) prone to develop a disease that models human IDDM. This procedure,which is known as oral tolerogenesis, is a possible means of preventingor retarding the complete development of insulitis and diabetes inindividuals in whom predictive immunoassay screening defines as being"at risk", i.e. in a presymptomatic incomplete stage of the disease.

Background

There is considerable interest among immunologists in the possibilitythat autoimmune disease in its earlier stages may be controlled bytreatments that can "rewire" the immune system so that a state ofdamaging immune reactivity to autologous (self) components can beswitched back to a normal state of tolerance to such components. This isdemonstrably achievable by delivery of antigen to mucosal tissues, andmore particularly the upper intestinal tract by the process of oraladministration by feeding the antigen, hence called oral tolerance. Oraltolerance has been shown to be effective in various experimental animalmodels of autoimmune disease (Weiner, 1997). The principle has beenapplied to the prevention of diabetes in the NOD mouse using theautoantigens insulin and GAD. However these experiments do not provideany teaching on optimal preparations of GAD for use in human therapy forwhich GAD purified from mammalian brain would be clearly unsuitable, andrecombinant GAD derived from currently used expression systems includingrabbit reticulocyte lysate or mammalian cells would be too low in yieldfor long term treatment of humans at risk for IDDM.

Protocol

The protocol for the study requires that three groups (n=10) of femaleNOD mice are fed GAD in amounts of 20 μg, 10 μg and 5 μg twice weeklyfor 30 weeks. During this time, animals are assayed weekly for increasedurinary glucose excretion and, when this is detected, blood glucose ismeasured. Appropriate control groups are included. The experimentascertains whether feeding GAD using a particular formulation retardsthe natural development of diabetes in NOD mice whereby some 70-80% ofuntreated females develop the disease.

Results

This experiment is still in progress. It has been ascertained thathybrid yeast GAD67/65 can be lyophilized and reconstituted as awater-soluble preparation suitable for mucosal administration to mice.Preliminary observations are that the feeding of GAD has no adverseeffects on mice. Moreover, there are no indications that oraladministration of GAD accelerates the development of insulitis in NODmice that are genetically predisposed to develop a disease thatreplicates many of the features of human IDDM.

Implications

The demonstration that orally administered hybrid yeast GAD67/65 canabrogate disease in the diabetes prone NOD mouse strain will haveimportant implications for interventional therapy at the preclinicalstage of human IDDM. In particular, the yeast system will provide forthe relatively large amounts of GAD that will be needed for human usewithout recourse to GAD prepared from ammalian sources, and yeast as acommon constituent of foods is a safe vehicle for preparation ofrecombinant GAD.

REFERENCES

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26 Richter, W., Y. Shiu and S. Baekkeskov. (1993). Autoreactive epitopesdefied by diabetes-associated human monoclonal antibodies are localizedin the middle and C-terminal domains of the smaller form of glutamatedecarboxylase. Proc. Natl. Acad. Sci. USA 90:2832-2836.

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38 Weiner, H. L. (1997). Oral tolerance: immune mechanisms and treatmentof autoimmune diseases. Immunology Today 18:335-343.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - <160> NUMBER OF SEQ ID NOS: 4                                        - - <210> SEQ ID NO 1                                                        <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer        - - <400> SEQUENCE: 1                                                         - - tggagctcat ggcgtcttcg accccatct         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 2                                                   <211> LENGTH: 23                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 2                                                         - - ttcgccggca gatctctagc aaa           - #                  - #                    23                                                                      - -  - - <210> SEQ ID NO 3                                                   <211> LENGTH: 29                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 3                                                         - - ccggaattca ccatggcgtc ttcgacccc         - #                  - #                29                                                                      - -  - - <210> SEQ ID NO 4                                                   <211> LENGTH: 63                                                              <212> TYPE: DNA                                                               <213> ORGANISM: Artificial Sequence                                           <220> FEATURE:                                                                <223> OTHER INFORMATION: Description of Artificial - #Sequence: Primer         - - <400> SEQUENCE: 4                                                         - - ccggaattct tagtggtggt ggtggtggtg agataaatct tgttcaaggc gt -             #tctatttc     60                                                                 - - ttc                  - #                  - #                  - #                 63                                                                __________________________________________________________________________

We claim:
 1. A hybrid glutamic acid decarboxylase (GAD) which comprisesan amino-terminal moiety which comprises amino acid 1 to amino acid90-105 derived from the human GAD67 isoform, said amino-terminal moietybeing linked directly, or indirectly via an additional amino acidsequence, with a carboxy-terminal moiety which comprises amino acid90-105 to amino acid 585 derived from the human GAD65 isoform.
 2. Ahybrid GAD according to claim 1, wherein said amino-terminal moietycomprises amino acid 1 to amino acid 95-101, of the human GAD67 isoform.3. A hybrid GAD according to claim 1, wherein said carboxy-terminalmoiety comprises amino acid 95-101 to amino acid 585, of the human GAD65isoform.
 4. A hybrid GAD according to claim 1, wherein theamino-terminal GAD67 moiety is linked directly to the carboxy-terminalGAD65 moiety.
 5. A hybrid GAD according to claim 1, wherein theamino-terminal GAD-67 moiety is linked indirectly to thecarboxy-terminal GAD65 moiety through a linker moiety of from 1 to about50 amino acid residues.
 6. A hybrid GAD according to claim 1, whereinthe amino-terminal GAD67 moiety is linked indirectly to thecarboxy-terminal GAD65 moiety through a linker of from 1 to about 20amino acid residues.
 7. A hybrid GAD according to claim 1, wherein theamino-terminal GAD67 moiety is linked indirectly to the carboxy-terminalGAD65 moiety through a linker moiety of from 1 to about 5 amino acidresidues.
 8. A hybrid GAD according to claim 1, which comprises aminoacid residues 1 to 101 of the human GAD67 isoform linked directly toamino acid residues 96 to 585 of the human GAD65 isoform.
 9. A hybridGAD according to claim 1, further comprising an additional moietycoupled at one or both ends, said additional moiety being selected froma group consisting of a glutathione-S-transferase moiety, aβ-galactosidase moiety and a hexa-His moiety.
 10. An isolated nucleicacid molecule comprising a nucleic acid sequence encoding a hybrid GADaccording to claim
 1. 11. An isolated DNA molecule comprising a nucleicacid sequence encoding a hybrid GAD according to claim
 1. 12. Arecombinant DNA cloning vector comprising a nucleic acid sequenceencoding a hybrid GAD according to claim
 1. 13. A host cell comprisingthe vector of claim
 11. 14. A host cell according to claim 11, which isa eukaryotic host cell.
 15. A host cell according to claim 14 which is ayeast cell.
 16. A host cell according to claim 15, which is a yeast cellselected from Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyceslactis, Hansenula polymorpha, or Schizosaccharomyces pombe.
 17. A methodfor the preparation of a hybrid GAD, which comprises expression of anucleic acid sequence encoding a hybrid GAD according to claim 1, in aeukaryotic host cell, and recovery of the hybrid GAD.
 18. A methodaccording to claim 17, wherein the eukaryotic host cell is a yeast cell.19. A method according to claim 18, wherein the yeast cell is selectedfrom Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces lactis,Hansenula potymorpha, or Schizosaccharomyces pombe.
 20. A method for thediagnosis and presymptomatic detection of IDDM in a human patient,characterised in that a hybrid GAD according to claim 1 is used todetect autoantibodies to GAD in serum taken from said patient.
 21. Acomposition, comprising a hybrid GAD according to claim 1, together withone or more pharmaceutically acceptable carrier and/or diluents.
 22. Acomposition according to claim 21 for mucosal administration.
 23. Acomposition according to claim 22 for oral administration.